Metal binding proteins and associated methods

ABSTRACT

Metal binding proteins, associated compositions and methods for their production and use are disclosed. The metal binding proteins included have amino acid sequences analogous to at least one metal binding protein, and conservative amino acid substitutions thereof from a brine shrimp ( Artemia ). Also provided are the associated nucleic acid sequences encoding metal binding proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending U.S. applicationSer. No. 11/510,485 filed Aug. 23, 2006 which is a division of U.S.application Ser. No. 10/797,748, now U.S. Pat. No. 7,135,605 which is adivision of U.S. application Ser. No. 09/948,495, now U.S. Pat. No.6,750,056, each of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to unique metal binding proteinshaving high binding affinity for heavy metals. More particularly, thepresent invention is directed to compositions including the unique metalbinding proteins and to associated methods of production and use wherereduction or recovery of heavy metals is desired.

BACKGROUND OF THE INVENTION

Metal recovery and metal remediation and the associated need forefficient and safe methods for clean up of metal waste is a continuingenvironmental and business concern due to the toxicity and potentialrisk to human health posed by metal contaminants, as well as theeconomic value of precious heavy metals. Indeed, as the discharge oftoxic wastes such as heavy metals from agricultural, industrial andother commercial operations continues, the need for effective, safe andlow-cost metal remediation methods increases. In a recent report by theU.S. EPA, metal contamination remains and historically has been a keyconcern at many contaminated sites (USEPA Work Assignment #011059, Mar.5, 1997, Contract #68-W5-0055). In addition, there are numerouspublished reports of damage to wildlife, livestock, plantlife as well asdanger to human health as a result of metal poisoning from contaminatedsoil or waste matter (Impact of Lead-Contaminated Soil on Public Healthby Xintaras, C. May 1992 at http://www.atsdr.cdc.gov/cxlead.html). Forexample, a primary concern to humans is the health hazard created bylead (Pb) contamination. Exposure to lead can occur through a variety ofmethods such as by ingestion of lead from food, water, soil, or eveninhalation of dust. Lead poisoning is extremely dangerous andpotentially fatal, with symptoms including seizures, mental retardationand behavioral disorders. Therefore, methods for metal remediation areextremely valuable both for their protection of our environment as wellas for protection from diseases.

Recovered metals from various waste, discard or recycling effortsprovide immense economic value as well as augmenting environmentalpollution control. Metal recovery can be from innumerable and variedsources such as from waste electronic devices (transistors, chips,transformers, bus bars, cathodes, and microprocessors, populatedcomputer circuit boards PCBs, motherboards). Costs associated withhazardous disposal of industrial waste in the absence of metalreclamation are enormous. Therefore, metal recycling or reuse of metalextracted from scrap or discarded metal-containing items not onlyreduces the volume and cost of metal waste requiring specializeddisposal and handling efforts, but the reclaimed metal can also beresold or reused to provide additional economic value.

Prior art attempts at treating metal contamination have traditionallyemployed cleanup technologies which consist primarily of physicallyremoving and then disposing of contaminated matter. These methodologiesare not only labor intensive and less efficient, but also carry a highexpense associated with removal and disposal of large or bulk quantitiesof contaminated waste. Metal contamination is especially difficult toremediate because unlike other types of waste such as chemical ororganic matter, metals cannot be directly destroyed or converted. Forexample, current technologies for remediating metal contaminated soilsconsist primarily of landfilling or soil excavation with physical orchemical separation of the metal contaminants. Treatment of contaminatedground water usually involves flushing, filtration or chemicalextraction to remove the contaminating metals. As a result, the cost ofsoil or ground water remediation is high, ranging in the hundreds tothousands of millions of dollars in projected five-year costs per site(U.S. EPA, 1993).

In addition, the risk to humans and the environment from heavy metalcontamination is not limited to soil or ground water, but also includesother sources such as industrial waste, sludge waste, wastewater,radionuclides (such as from research and medical waste) and miningwaste. Depending on the physical and chemical form of the metalcontaminant to be removed, as well as the cost-benefit analysis for aparticular remediation approach, which of the existing technologies isbetter suited for a particular site will vary. However, due to the highcost of traditional cleanup technologies, there still remains a greatneed for a less-expensive, safe and effective heavy metal recovery andcleanup technology.

There are some technologies currently available for the recovery orremediation of heavy metal contaminated waste. In general, thesetechnologies combine one or more of the following general approaches:isolation, immobilization, toxicity reduction, physical separation orextraction of metal contamination from a waste product. Isolationtechnologies utilize a containment strategy in an attempt to confine acontaminated site or area so as to prevent further spread of the toxicmetal waste. Immobilization technologies reduce the mobility of metalcontaminants and include systems which provide an impermeable barrier toseparate underlying layers of soil (containing the metal contaminants)from the topsoil layer. Also used are physical barriers which restrictthe flow of uncontaminated groundwater through a contaminated site.Additionally, there are toxicity reduction processes which generally usechemical or biological techniques to decrease the toxicity or mobilityof metal contaminants. Included in toxicity reduction processes arebiological treatment technologies, which apply newer biotechnicalapproaches.

Metal remediation is a relatively new application of biologicaltreatment technologies and includes processes such as bioaccumulation,phytoremediation, phyotextraction, and rhizofiltration. All of thesebiological-treatments use certain plants and microorganisms to remediatemetals through either adsorption, absorption, or concentration ofcontaminating metal ions. For example, in bioaccumulation, plants ormicroorganisms actively take up and accumulate metals from contaminatedsurroundings.

In phytoremediation, specific plants that have developed the ability toselectively remove metal ions from soil are used. Such plants includecertain “hyperaccumulator” species such as the alpine pennycrass plant,which is capable of accumulating metals at levels of 260 times greaterthan most plants before showing toxicity symptoms. Most hyperaccumulatorplants, however, are very slow growing and have specific growthrequirements. Some of these growth requirements are not conducive to theuse of these plants at sites or in situations where metal recovery orremediation is needed. Furthermore, there are very few plant speciesknown or available for recovery or remediation use. Therefore, given thepersistent and high incidence of metal contamination at environmentaland waste sites (.about.75% of Superfund Sites contain metal ions as aform of contamination, U.S. EPA, 1996), more efficient methods andapproaches for removing heavy metals from contaminated sources are stillneeded.

More recently, in an attempt to meet these needs, biotechnologicalapproaches have been employed as an alternative strategy to metalrecovery and remediation. Included in these biotechnology approaches arethe use of tobacco plants that have been manipulated to expressmetallothionein genes (Maiti et al., 1991). Metallothioneins (MTs) aresmall metal binding proteins ubiquitously distributed throughout theanimal kingdom. They have high metal binding affinities and are believedto be important in controlling the intracellular levels of free metalions. However, little else is known about their function or biologicalpurpose. MTs were first discovered in 1957 in horse tissue. Since then,they have been identified in species ranging from fungi and shellfish tomice and humans.

The structural features of MTs include a high cysteine composition andlack of aromatic amino acids. The cysteine residues are responsible forthe protein's high affinity metal ion binding capabilities. In general,prior art MTs have a high degree of amino acid sequence similarity.However, the proteins or known gene sequences encoding the prior artproteins have been used primarily in either the research setting or indisease treatment methodologies.

Accordingly, one of the objects of the present invention is to providenovel metal binding proteins and associated methods for theirproduction. This technology would allow for the efficient, costeffective, safe and simple removal of heavy metals from environmentalwaste or other materials contaminated with heavy metal.

SUMMARY OF THE INVENTION

Prior art metallothionein (MT) proteins are generally about 60-68 aminoacid residues in size and have a high degree of sequence conservationamong the different species. Whereas this high degree of sequenceconservation and similarity contributed greatly to the ease of discoveryof those MT genes, the novel metal binding proteins of the presentinvention differ substantially in sequence and were, therefore, muchmore difficult and required greater perseverance to obtain.

Unlike known prior art MTs, MTs from brine shrimp (Artemia) are muchsmaller in size (about 48 amino acid residues) and have distinctlyunique amino acid and DNA sequences. As a result of these divergences insequence from prior art MTs, prior to the present invention, the novelmetal binding proteins of the present invention were extremely difficultto obtain and the nucleic acid sequences encoding these novel metalbinding proteins unknown. The novel metal binding proteins of thepresent invention are capable of high capacity and high affinity metalbinding. This makes them particularly suitable for use in pollutioncontrol, metal recycling, metal mining and other metal recovery andmetal remediation technologies.

These and other objects are achieved by the compositions and methods ofthe present invention which provide for the efficient and reliablesequestration of heavy metals from a variety of sources. The novel metalbinding proteins of the present invention can be expressed and producedeasily for purposes such as metal remediation, metal recycling, metalmining or other types of processes where binding of one or more heavymetals is desired.

In accordance with the teachings of the present invention, novel metalbinding proteins are provided. The invention includes at least onesubstantially purified metal binding protein having an amino acidsequence analogous to at least one metal binding protein sequence frombrine shrimp (Artemia). A substantially purified metal binding proteincan include an amino acid sequence such as:

MET ASP CYS CYS LYS ASN GLY CYS THR CYS ALA PRO ASN CYS LYS CYS ALA LYSASP CYS LYS CYS CYS LYS GLY CYS GLU CYS LYS SER ASN PRO GLU CYS LYS CYSGLU LYS ASN CYS SER CYS ASN SER CYS GLY CYS HIS STOP [SEQ ID NO: 2]; METASP CYS CYS LYS ASN GLY CYS THR CYS ALA PRO ASN CYS LYS CYS ALA LYS ASPCYS LYS CYS [SEQ ID NO: 4];

and sequences incorporating one or more conservative amino acidsubstitutions of SEQ ID NO: 2 or SEQ ID NO:4. It should be noted thatwhile the present invention will be discussed in the context of metalrecovery and metal remediation, the present invention is readilyapplicable to many other uses where removal, recovery or simply bindingof heavy metals is desired.

The metal binding proteins of the present invention also include afamily of metal binding proteins having multiple isomeric forms.Accordingly, the family of metal binding proteins includes at least 5isomeric forms of metal binding proteins. Any and all of these metalbinding protein isomers are suitable for use in removal or recovery ofheavy metals. The “isomers” of the present invention have the requisitestructural features that classify them as metal binding proteins. Thesefeatures include their high cysteine content, which confers their metalbinding capacity. Therefore, the metal binding proteins of the presentinvention, including their isomeric forms, can be expressed and easilyproduced for purposes such as metal remediation, metal recycling, metalmining or other types of processes involving metal binding.

The novel metal binding proteins of the present invention also havecharacteristics that further enhance their use in the methods of thepresent invention and which further distinguish them from the prior art.These advantageous characteristics also render the novel metal bindingproteins and associated methods particularly useful in a wide variety ofmetal recovery and metal remediation settings. For example, the metalbinding proteins are capable of heavy metal binding under a range ofconditions such as under moderate to high temperature conditions. Metalbinding activity occurs from about 4° C. to about 100° C. Depending on aparticular application or operation in which a metal binding protein ofthe present invention is to be implemented, a particular temperaturerange may be preferred. Therefore, in accordance with the presentinvention, the suitable range of temperatures include anywhere fromabout 4° C. to about 100° C. This range of temperature conditions whichis incompatible with some prior art methodologies, renders oursubstantially purified metal binding proteins more versatile andpreferable for use in metal recovery, metal remediation or otherprocesses requiring heavy metal binding. Additionally, the metal bindingproteins of the present invention are able to bind metal in a variety ofchemical conditions. For example, metal binding activity occurs fromabout pH 4.0 to about 10.0. Bound metal ion(s) can be disassociated orremoved from a metal binding protein of the present invention bylowering the pH to about 1.0. An exemplary method comprises slowlyincreasing the pH to about 7.0 in the presence of a reducing agent, suchas dithiothreitol (DTT) for example. This reestablishes the metalbinding activity of the metal binding protein and, therefore, the metalbinding proteins can be reused if desired.

In further accordance with the teachings of the present invention,isolated nucleic acids encoding the metal binding proteins are provided.These isolated nucleic acids encode metal binding proteins having aminoacid sequence analogous to at least one metal binding protein sequencefrom a brine shrimp (Artemia). An isolated nucleic acid of the presentinvention can include a DNA sequence such as:

[SEQ ID NO: 1] 2 5′-ATG GAC TGC TGC AAG AAC GGT TGC ACC TGT GCC CCA AATTGC AAA TGT GCC AAA GAC TGC AAA TGC TGC AAA GGT TGT GAG TGC AAA AGC AACCCA GAA TGC AAA TGT GAG AAG AAC TGT TCA TGC AAC TCA TCT GGT TGT CACTGA-3′.

Alternatively, an isolated nucleic acid of the present invention caninclude minimal DNA sequences which are sufficient to allow translationof a functional metal binding protein. A DNA sequence encoding afunctional metal binding protein of the present invention need notcomprise the entire native metal binding protein gene sequence but canbe just those portions or regions of SEQ ID NO:1 that confer binding toheavy metals. For example, the present invention can include a DNAsequence comprising:

[SEQ ID NO: 3] 3 5′-ATG GAC TGC TGC AAG AAC GGT TGC ACC TGT GCC CCA AATTGC AAA TGT GCC AAA GAC TGC AAA TGC-3′.

Additionally, the present invention also includes DNA having at least80% or more sequence identity to a DNA molecule having the sequence ofSEQ ID NO:1 or a DNA molecule having the sequence of nucleotide residues1 to 66 of SEQ ID NO:1.

The isolated nucleic acids of the present invention also include nucleicacids encoding any and all of the isomeric or alternative forms of themetal binding proteins disclosed. Additionally, the isolated nucleicacids of the present invention need not comprise entire coding sequencesof an MT isomer, but can include nucleic acid sequences encoding domainsor portions of a coding sequence encoding an MT isomer, such as, forexample, the functional or metal binding regions of the metal bindingprotein isomers of the present invention.

In further accordance with the teachings of the present invention, thenovel metal binding proteins can be utilized as a naked composition orcan be provided in association with a support, substrate, or otherdelivery system to aid in either the dispersal, handling, packaging orfunction of the metal binding proteins in metal recovery, metalremediation or metal binding processes as disclosed herein. Therefore,any of the metal binding proteins of the present invention can becoupled to a support such as a membrane or filter through which metalcontaining fluids are brought into contact.

The present invention is particularly well suited for use in metalrecovery, metal remediation or metal recycling processes and methods.These methods include contacting a metal binding protein of the presentinvention having an amino acid sequence analogous to at least one metalbinding protein sequence from brine shrimp (Artemia) with a substrate ormaterial having a concentration of at least one heavy metal in order tobind the metal to the metal binding protein; and then separating thebound metal from the substrate or material.

For example, the methods of the present invention are useful inconnection with the treatment of any substance having a concentration ofat least one heavy metal. As will be appreciated by those skilled in theart, such heavy metal containing substances can be any environmental orindustrial material such as ground water, drinking water, contaminatedsoil, waste, or the like, containing a concentration of metal.Similarly, the methods of the present invention are equally useful intreating industrial or municipal wastes containing metals that aredesirable to remove. This broad utility makes the compositions andassociated methods of the present invention particularly useful in awide variety of circumstances.

In further accordance with the novel teachings of the present invention,expression systems producing the novel metal binding proteins of thepresent invention are provided. These novel metal binding proteinsproduced by the disclosed expression systems include the metal bindingproteins having amino acid sequences analogous to at least one bindingprotein sequence from a brine shrimp (Artemia), as previously discussed.These expression systems include systems for the production ormanufacturing of these compositions which can function in larger scalecommercial or industrial plants, as well as in smaller scale,site-specific applications. Also included within the teachings of thepresent invention are expression systems of live or living productionentities such as modified organisms and host cells. These includetransgenic plants, transgenic animals and bacteria, and other modifiedorganisms which have been genetically engineered to produce the novelmetal binding proteins of the present invention.

Accordingly, the present invention provides unique, relatively smallmetal binding proteins having unique properties and sequences distinctfrom those of known prior art metal binding proteins. Furthermore, thenovel metal binding proteins of the present invention retain highbinding affinity for heavy metals in a variety of conditions, makingthem particularly useful in situations where removal or recovery ofheavy metals from a substrate or any metal containing or metalcontaminated source is desired. The novel metal binding proteins and theassociated methods of the present invention provide for the efficient,cost effective, and safe removal and recovery of heavy metals from awide variety of substrates.

The following Detailed Description provides additional enablingdisclosure of the present invention and will make apparent to thoseskilled in the art additional features and advantages thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution profile of exemplary metal binding proteins of thepresent invention illustrating co-elution of metal binding proteins withthe heavy metal, zinc.

FIG. 2 is a map of an exemplary cloning cassette containing the genesequence of the metal binding gene, in accordance with the teachings ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Known metal binding proteins (MTs) that have been isolated from variousspecies such as humans, rodents, bacteria, crabs, chickens, etc. . . .and are known to have very similar structural characteristics such assimilar size (˜6.0-6.8 kDa), high amino acid sequence conservation, anda high percentage of cysteine residues in the proteins' total amino acidcompositions. It is the cysteine composition of these known MTs thataccounts for the protein's binding affinity for heavy metals such aszinc, copper, cadmium, mercury, cobalt, lead, nickel, platinum, silverand gold.

The novel metal binding proteins of the present invention are distinctfrom all other known MTs because the proteins of the present inventionhave uniquely different amino acid sequences and DNA sequences. Plus,the proteins of the present invention are much smaller in size whencompared to known MTs from other species. Yet, in spite of thesedifferences, the proteins of the present invention retain the ability tobind metals with unexpectedly high affinities. This unusual sequencedivergence also made the novel metal binding proteins of the presentinvention much more difficult to isolate and characterize compared toprior art MTs. For example, the novel metal binding proteins of thepresent invention are particularly useful in the efficient and costeffective removal of heavy metals from industrial, municipal, andenvironmental waste. This use of these metal binding proteins for metalremediation is of particular value for pollution control and isparticularly applicable to the removal of heavy metals from groundwater, contaminated soil, drinking water or any material having heavymetal contamination.

Alternatively, the novel metal binding proteins of the present inventionare also very useful in the recovery of metals, particularly preciousmetals. For example, the metal binding proteins of the present inventioncan be used in metal mining processes for the isolation and removal ofprecious metals such as gold, platinum and silver. Doing so eliminatesthe need to use other toxic metals such as mercury in the final stagesof metal purification from ore. These same novel techniques can beutilized to recover such metals from industrial or municipal waste. Withthe ever increasing use of disposable and other electronic devices, suchwaste sources are increasingly full of such metals, making recovery aworthwhile endeavor.

In either situation, due to the smaller size of these proteins and theuniquely specific sequence information provided herein, the novel metalbinding proteins of the present invention can be isolated easily andefficiently from natural sources or synthetically produced as disclosedherein for use in metal recovery, metal mining, metal recycling, metalremediation, pollution control or any process including metalsequestering. Therefore, the novel metal binding proteins and associatedmethods of the present invention provide a versatile, easily produced,efficient and reliable resource for use in any process having a metalbinding aspect.

The novel metal binding proteins of the present invention were firstisolated from brine shrimp (Artemia). They are a family of metal bindingproteins which are referred to as “isomers”. Analysis of these proteins'unique amino acid compositions showed each isoform to be essentiallyequivalent. At least five individual isoforms have been identified inaccordance with the teachings of the present invention. Unlike MTs fromother organisms which share a high degree of sequence homology orsimilarity, the novel metal binding proteins of the present inventionhave unexpectedly different structural characteristics but possess ahigh degree of sequence homology to one another. These distinct sequencecharacteristics of these proteins of the present invention preventedtheir earlier isolation and characterization.

After numerous unsuccessful attempts at isolating a gene encoding brineshrimp MT using conventional approaches based upon prior art teachingsand MT sequence information, the following techniques were utilized toprovide nucleic acid sequence encoding a novel metal binding protein ofthe present invention. First, a sample of metal binding protein frombrine shrimp (Artemia) was isolated and purified. N-terminal amino acidsequence analysis was performed on this metal binding protein-containingsample. This amino acid sequence analysis indicated that the position ofthe first six cysteine residues of the target brine shrimp (Artemia)metal binding protein was conserved when compared to equine and humanMTs, indicating the importance of these amino acid residues in theprotein's metal binding function.

Using this N-terminal amino acid sequence information, oligonucleotideprimers corresponding to the N-terminal amino acid sequence wereconstructed as known in the art. These oligonucleotide primers were usedto PCR amplify potential candidates for a MT gene sequence encoding atleast one of the target metal binding proteins from brine shrimp(Artemia). The PCR product was purified using QiaPrep spin columns(Qiagen, Inc.) and cloned into the TA cloning vector CR2.1 (InVitrogen)using the manufactures' protocol. Electrocompetent E. coli (Sure Shotcells from InVitrogen) were transformed with the recombinant vector andplated onto LB agar plates containing ampicillin (100 μg/ml) and 1%glucose. The plates were placed at 37° C. overnight. Individual colonieswere picked and used to inoculate 5 ml of LB broth supplemented withampicillin and 1% glucose. The cultures were incubated overnight in arotary incubator at 37° C. Plasmid was isolate from 2 ml of the cellsuspension using QiaPrep spin columns as per the manufacture's protocol(Qiagen). The plasmid was then sequenced on a Li Cor 4200 L using theM13 universal forward and reverse primers. Once verified and determinedto be a sequence encoding a metal binding protein, the brine shrimp MTgene was subcloned into the bacterial expression vector pTMZ. Based uponthe identified MT encoding sequence, the amino acid sequence of thefirst novel metal binding proteins of the present invention wasdetermined. FIG. 1 details an exemplary elution profile utilizing anexemplary metal binding protein of the present invention. This profilewas obtained utilizing the following exemplary protocol.

E. coli (Strain ER 2566) were transformed with a plasmid expressionvector containing the MT gene sequence [SEQ ID NO: 1] in (pTMZ).Bacteria were grown in LB broth containing 1% glucose at 37° C. to anA₆₀₀ of 0.60. The bacterial cells were collected and resuspended in LBbroth containing 0.1% glucose and incubated for 45 minutes at the sametemperature. Isopropyl b-D-thiogalactopyranoside (IPTG) was added to afinal concentration of 0.1 mM. The bacterial cells were incubated forabout 16 hours. Non-transformed bacteria were used as controls. Thecells were collected by centrifugation and sonicated in 10 mM Tris, pH8.0, 5 mM DTT and 0.5 mM PMSF. The homogenate was centrifuged at150,000.×g for 1 hour at 4° C. The supernatant was collected andincubated with 2 μCi of ¹⁰⁹Cd at room temperature. The radiolabeledsupernatant was then applied to a G-50 molecular exclusion column andeluted with 50 mM Tris, pH 8.0. Five ml fractions were collected andassayed for radioactivity (CPM) and zinc (PPB), the zinc being anendogenous metal that associates with the exogenous metal bindingprotein expressed by the transformed bacteria. Each fraction elutingfrom the column was assayed for Zn by ICPMS (Inductively Coupled PlasmaMass Spectroscopy). The DNA sequence that encodes a functional metalbinding protein, such as [SEQ ID NO: 3], may also be utilized, asprovided and disclosed by the teachings of the present invention.

Therefore, the present invention provides substantially purified metalbinding proteins having amino acid sequence to at least one metalbinding protein sequence from a brine shrimp (Artemia). The term“substantially purified”, as used herein, refers to nucleic acids, aminoacids or proteins that have been removed from their natural environment,isolated or separated and are at least 60% free, preferably 75% free, to90% or more free from other components with which they are naturallyassociated.

A substantially purified metal binding protein in accordance with theteachings of the present invention has an amino acid sequence analogousto:

[SEQ ID NO: 2] MET ASP CYS CYS LYS ASN GLY CYS THR CYS ALA PRO ASN CYSLYS CYS ALA LYS ASP CYS LYS CYS CYS LYS GLY CYS GLU CYS LYS SER ASN PROGLU CYS LYS CYS GLU LYS ASN CYS SER CYS ASN SER CYS GLY CYS HIS STOP.

Also within the scope of the present invention are substantiallypurified metal binding proteins that are variants of the sequence of theabove SEQ ID NO: 2 that preserve the protein's metal binding affinity.In particular, conservative amino acid substitutions within the scope ofthe present can include any of the following: (1) any substitution ofisoleucine for leucine or valine, leucine for isoleucine, and valine forleucine or isoleucine; (2) any substitution of aspartic acid forglutamic acid and of glutamic acid for aspartic acid; (3) anysubstitution of glutamine for asparagine and of asparagine forglutamine; and (4) any substitution of serine for threonine and ofthreonine for serine.

A “conservative amino acid substitution” as used herein, refers toalteration of an amino acid sequence by substituting an amino acidhaving similar structural or chemical properties. Those skilled in theart can determine which amino acid residues may be substituted, insertedor altered without the metal binding properties of the proteins of thepresent invention.

Other substitutions can also be considered conservative, depending uponthe environment of the particular amino acid. For example, glycine (G)and alanine (A) can be interchangeable, as can be alanine and valine(V). Methionine (M), which is relatively hydrophobic, can beinterchanged frequently with leucine and isoleucine, and sometimes withvaline. Lysine (K) and arginine (R) are interchangeable in locations inwhich the significant feature of the amino acid residue is its chargeand the different pK's of these two amino acid residues and where theirdifferent sizes are not significant. Still other changes can beconsidered “conservative” in particular environments, as known in theart.

For example, if an amino acid on the surface of a protein is notinvolved in a hydrogen bond or salt bridge interaction with anothermolecule, such as another protein subunit or a ligand bound by theprotein, negatively charged amino acids such as glutamic acid andaspartic acid can be substituted with positively charged amino acidssuch as lysine or arginine and vice versa. Histidine (H), which is moreweakly basic than arginine or lysine, and is partially charged atneutral pH, can sometimes be substituted for these more basic aminoacids as well. Additionally, the amides glutamine (Q) and asparagine (N)can sometimes be substituted for their carboxylic acid homologues,glutamic acid and aspartic acid.

The novel metal binding proteins of the present invention, and theirassociated methods of production and use, are a family of metal bindingproteins having multiple isomeric forms. As a result, the presentinvention includes at least 5 isomeric forms of metal binding proteinssuitable for use in removal or recovery of heavy metals. An isomer isone of two or more compounds that have the same chemical composition butdiffer in structural form. The “isomers” of the present invention havethe requisite structural features that classify them as metal bindingproteins. These features include their high cysteine content, whichconfers their metal binding capacity. The isomers differ by two or moreamino acid residues, resulting in different pl's for the individualisomer. This pl difference allows easy separation and characterizationof the isoforms. Therefore, the metal binding proteins of the presentinvention can be expressed and produced efficiently and with ease.

In addition to their metal binding properties, the metal bindingproteins of the present invention also exhibit features which renderthem particularly useful in a wide variety of metal recovery and metalremediation settings. For example, these novel metal binding proteinsare capable of heavy metal binding under a range of conditions such asunder moderate to high temperature conditions. The novel metal bindingproteins are capable of heavy metal binding at room temperature andtherefore particularly ideal for many applications. The novel metalbinding proteins are also capable of heavy metal binding within a widetemperature range such as, for example, a temperature range of about 4°C. to about 100° C. Those skilled in the art will appreciate thatdepending on a particular application or operation in which the metalbinding proteins are to be utilized, a particular temperature range maybe preferred for practical or economic reasons. For example, it may bemore practical to use the metal binding proteins “on-site” or at thelocation of an environmental contamination (which would dictate thatparticular temperature range that can be obtained within availablecosts). On the other hand, more effective metal extraction on certainsubstrates may be achieved by use of the metal binding proteins of thepresent invention under relatively high temperature conditions.Therefore, in accordance with the teachings of the present invention, asuitable range of temperatures for practicing the present inventionincludes a range of about 4° C. to about 100° C. This range oftemperature conditions makes the metal binding proteins of the presentinvention more versatile and useful.

In further accordance with the teachings of the present invention, themetal binding proteins can be utilized as a naked composition or inassociation with a substrate or dispersal means to aid in either thedispersal, handling, packaging or function of the metal binding proteinin metal recovery, metal remediation or metal binding processes. Suchmetal binding proteins are particularly useful in metal recovery, metalremediation and metal binding processes because they can be more easilyand safely used as compared to other methodologies, such as chemicalextraction, which exposes the user to toxic or other potentiallydangerous types of chemicals.

A variety of supports to aid in the handling or dispersal of the novelmetal binding proteins can be used and include solid supports, matrices,membranes, semi-permeable membranes, powders, devices, apparatuses,liquids, formulations, and other materials. It should be noted, that anadditional characteristic feature of the novel metal binding proteinsare that they are also capable of reversible heavy metal binding. Forexample, bound metals can be eluted off or away from the metal bindingproteins using acidic conditions or by instantaneous exchange reactionsor inorganic chelators. For example, during incubation of a metalbinding protein with radioactive Cd, the ¹⁰⁹Cd metal exchanges forendogenous metal bound to the metal binding protein. At about pH 1.0,the metal is released from the protein. Bringing the pH of the solutionup to about pH 8.0 regenerates the metal binding activity of theprotein. Therefore, due to the reversible binding characteristics of thenovel metal binding proteins, the present invention also providescompositions, formulations, powders, liquids, devices or apparatusescomprising the substantially purified metal binding proteins which canbe utilized more than once, or which can be reused.

Turning now to an exemplary discussion of the genetic engineering of thenovel metal binding proteins of the present invention, a nucleotidesequence for one of the isoforms of a metal binding protein from a brineshrimp (Artemia) was identified, as discussed above. Generally, theisolation process comprises: (1) preparation of one or more sample(s)containing nucleic acids from brine shrimp (Artemia); (2) isolation oftotal RNA from Artemia; (3) preparation of cDNA from the total RNA; (4)amplification of metal binding protein gene sequences; and (5) cloning,sequencing and verification of an isolated nucleic acid sequence as ametal binding protein gene (MT) from brine shrimp (Artemia).

The above procedure yielded the entire coding sequence for one of themetal binding protein genes, MT. This sequence is:

[SEQ ID NO: 1] 5′-ATG GAC TGC TGC AAG AAC GGT TGC ACC TGT GCC CCA AATTGC AAA TGT GCC AAA GAC TGC AAA TGC TGC AAA GGT TGT GAG TGC AAA AGC AACCCA GAA TGC AAA TGT GAG AAG AAC TGT TCA TGC AAC TCA TGT GGT TGT CACTGA-3′

Therefore, the invention also provides one or more nucleic acidsequences encoding a substantially purified metal binding protein havingamino acid sequence analogous to at least one metal binding proteinsequence from brine shrimp (Artemia). The nucleic acid sequences includethe sequence of SEQ ID NO: 1; a sequence complementary to SEQ ID NO: 1;or a sequence complementary to SEQ ID NO: 1 with no greater than about a15% mismatch under stringent conditions. Preferably, the degree ofmismatch is no greater than about 5%; most preferably the mismatch is nogreater than about 2%.

Alternatively, an isolated nucleic acid can comprise the minimal DNAsequences sufficient to allow translation of a functional metal bindingprotein. A functional metal binding protein need not be the entirenative metal binding protein but can be just those portions or regionsof SEQ ID NO:1 that encodes a protein capable of binding to heavymetals. Therefore, the invention also includes isolated nucleic acidsincluding DNA having at least 80% sequence identity to a DNA moleculehaving the sequence of nucleotide residues 1 to 66 of SEQ ID NO: 1.

Also within the present invention is a nucleic acid sequence encodingany one of the novel metal binding proteins of the present invention.Such novel metal binding proteins can have molecular weight of about5,800 daltons and are able to bind with high affinity to heavy metalions such as zinc, copper, cadmium, mercury, cobalt, lead, nickel,platinum, silver and gold, etc . . . . The novel metal binding proteinsinclude therein an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 2 and sequences incorporating one or moreconservative amino acid substitutions thereof wherein the conservativeamino acid substitutions are any of the following: (1) any ofisoleucine, leucine and valine for any other of these amino acids; (2)aspartic acid for glutamic acid and vice versa; (3) glutamine forasparagine and vice versa; and (4) serine for threonine and vice versa.Alternative nucleic acid sequences can be determined using the standardgenetic code; the alternative codons are readily determinable for eachamino acid in this sequence.

It should be noted, that while the isolated nucleic acids providedherein can be used to produce or express novel metal binding proteins,they are also particularly useful for isolation and identification ofadditional metal binding protein genes encoding the novel metal bindingproteins of the present invention. For example, using the strategy,exemplary methods and nucleic acid sequences provided herein, DNAsequences encoding any of the metal binding protein isomers can beobtained. Therefore, the present invention includes nucleic acidsencoding any and all of the isomeric or alternative forms of the metalbinding proteins of the present invention. Additionally, isolatednucleic acids need not comprise entire coding sequences of an MT isomer,but include nucleic acid sequences encoding domains or portions of acoding sequence encoding an MT isomer, such as the functional or metalbinding regions of the metal binding protein isomers of the presentinvention.

Construction and isolation of nucleic acid sequences according to thepresent invention can be accomplished by techniques well known in theart, including solid-phase nucleotide synthesis, the polymerase chainreaction (PCR) technique, reverse transcription of DNA from RNA, the useof DNA polymerases and ligases, and other techniques. Using an aminoacid sequence encoding a metal binding protein or portion of a metalbinding protein of the present invention, the corresponding nucleic acidsequence can be constructed according to the genetic code as known inthe art.

Another aspect of the invention is a vector comprising a nucleic acidsequence according to the present invention operatively linked to atleast one control sequence that controls the expression or regulation ofthe nucleic acid sequence. Such control sequences are well known in theart and include operators, promoters, enhancers, promoter-proximalelements and replication origins. The techniques of vector construction,including cloning, ligation, gap-filling, the use of the polymerasechain reaction (PCR) procedure, solid-state oligonucleotide synthesis,and other techniques, are all well known in the art and need not bedescribed further here. The vectors of the present invention areparticularly useful in producing the novel metal binding proteins eitherby modified organisms, host cells or other types of expression systems.

Therefore, another aspect of the present invention is a host cell ormodified organism transfected with a vector according to the presentinvention. Among the host cells that can be used are bacteria, plants,algae, shrimp, fish, or any organism suitable for genetic modificationto produce a novel metal binding protein of the present invention.

Transfection, also known as transformation, is performed using standardtechniques appropriate to the host cell or organism used. Suchtechniques are described, for example, in Sambrook et al., supra.

Turning now to uses for the novel metal binding proteins of the presentinvention. These include pollution control applications of the metalbinding proteins such as metal remediation, pollution control, metalrecycling or metal mining. For example, the novel metal binding proteinscan be used to reduce the concentration of heavy metals in anenvironmental substance. The substance can be a fluid, such as groundwater, sludge, waste-water and the like. Additionally, the novel metalbinding proteins can be incorporated into one or more compositions ordevices used for pollution control. For example, the novel metal bindingproteins can be applied on site in the form of a flocculent or powder,or can be used in treatment plants as part of a membrane filtration orother type of solid support device used for removal of heavy metal froma contaminated substrate.

The novel metal binding proteins used in these metal binding processescan be provided as a product purified from its natural source or can beproduced by bioengineering techniques. For example, the novel metalbinding proteins can be produced by transgenic or modified organisms.Modified organisms include transgenic animals, bacteria or plants. Forexample, a modified plant can be a transgenic tobacco plant whose genomehas been genetically altered to express one or more novel metal bindingprotein of the present invention. A modified organism can also include aplant or biomass that is capable of growing at or within contaminatedsites where metal remediation is desired. Extraction of metalcontaminants by the modified organisms also concentrates the toxicmetals from the contaminated site. This provides the additionaladvantage of converting the heavy metals to a smaller quantity as wellas providing final product that is more easily and safely handled fordisposal or further processing.

Methods for reducing the concentration of heavy metals in a substrateinclude contacting a novel metal binding protein of the presentinvention with a substrate having heavy metals. For example, a metalbinding protein having an amino acid sequence analogous to at least onemetal binding protein sequence from brine shrimp (Artemia) can becontacted with a substance having a concentration of at least one heavymetal to bind the heavy metal to the metal binding protein.Subsequently, the bound heavy metal can be separated from the substrate,reducing the concentration of heavy metals in the original substrate.

As mentioned previously, an additional advantageous feature of the novelmetal binding proteins of the present invention include their ability torelease bound heavy metals using acid extraction, inorganic chelators,and/or exchange reaction technologies. This allows the user, if desired,to elute bound heavy metals off the novel metal binding proteins. Oncethe heavy metals are eluted off the metal binding proteins of thepresent invention, the metal binding proteins can be regenerated (orrecycled) for additional uses in metal extraction. Therefore, theinvention also provides methods for reducing the concentration of heavymetals in a substrate using reusable compositions, devices andapparatuses comprising the novel metal binding proteins.

Metal binding proteins of the present invention, when used in methodsfor reducing the concentration of a metal ion in a substrate can beprovided in such a way as is appropriate for the particular use,situation, mode of administration or environment in which the metalbinding proteins are to be used. For example, when used in metalremediation, or in pollution control, the metal binding proteins can becoupled to a support, such as a powder and used, for example, as aflocculent to provide a convenient and efficient means of dispersing themetal binding proteins.

Alternatively, the metal binding proteins can be provided coupled to amembrane, a semi-permeable membrane, a filter, or any other meansappropriate for allowing sufficient exposure of the novel metal bindingproteins to the heavy metal containing substrate so as to bind orsequester the heavy metals from the substrate. A membrane or filtercomprising the novel metal binding proteins provides a particularlyefficient means of treating ground water or waste water, as contaminatedwater can be purified by passage through the membrane or filter withoutfurther clean up as is required in chemical extraction processes.Coupling the novel metal binding proteins to a support or supportingmatrix also affords easier handling of the metal binding proteinsespecially when used in large scale or industrial applications.

Use of the metal binding proteins of the present invention are notlimited only to those methods where removal of heavy metals is desired,but can also include methods where recovery or concentration of heavymetals in a substance is to be achieved. For example, the novel metalbinding proteins can be used for metal-mining, such as in the recoveryof precious metals including gold, platinum and silver, or can be usedto concentrate metals in hazardous conditions, such as hazardous wastecontaining radioactive metals. Such hazardous metal waste can resulteither from numerous research, commercial or industrial uses.

Use of the novel metal binding proteins in concentrating radioactivemetals from waste also reduces the amount or quantity of hazardous wasteto be disposed of. Reducing the quantity of hazardous metal waste alsoreduces the level of radioactivity to which certain individuals areexposed. For example, individuals working with or near materialscontaining hazardous heavy metals will have a reduced overall exposureto radioactive metal waste due to the decreased quantity or volume ofradioactive waste requiring human handling. This provides an additionaladvantageous aspect of using the novel metal binding proteins of thepresent invention in metal binding processes.

Methods for reducing the concentration of heavy metals in a substanceinclude producing the novel metal binding proteins in a modifiedorganism. Modified organisms include, for example, transgenic organismsor transgenic hosts. For example, hosts or organisms such as shrimp,plants, bacteria, or algae can be modified using molecular and geneticengineering techniques well known in the art. Using these techniques,which are described for example, in Sambrook et al., Molecular Cloning:A Laboratory Manual (New York: Cold Spring Harbor Press, 2001); Ausubelet al. Current Protocols in Molecular Biology (Wiley IntersciencePublishers, 1995); US Dept Commerce/NOAA/NMFS/NWFSC Molecular BiologyProtocols (URL:http://research.nwfsc.noaa.gov/protocols-.html); orProtocols Online (URL:www.protocol-online.net/molbio/index.htm),organisms whose genome are modified so as to result in expression of anovel metal binding protein are provided. Novel metal binding proteinsof the present invention include metal binding proteins having an aminoacid sequence analogous to at least one metal binding protein sequencefrom a brine shrimp (Artemia). Modified organisms can be made and usedto produce these novel metal binding proteins, and the novel metalbinding proteins useful in the methods provided herein.

A modified organism producing a novel metal binding protein of thepresent invention includes a modified organism producing at least onemetal binding protein having an amino acid sequence substantiallysimilar to a metal binding protein from a brine shrimp (Artemia). Amodified organism also includes an organism producing a metal bindingprotein having an amino acid sequence substantially similar to SEQ IDNO: 2 or conservative amino acid substitutions thereof.

Alternatively, production or expression of the novel metal bindingproteins of the present invention from modified organisms is not limitedto genomic expression of the novel metal binding proteins, but alsoincludes epigenetic expression of the novel metal binding proteins fromthe modified organisms. Methods and techniques for obtaining epigeneticexpression from a modified organism include, for example, adenoviral,adeno-associated viral, plasmid and transient expression techniqueswhich are known in the art.

When dealing with genes from eukaryotic organisms, it is preferred touse cDNA, because the natural gene typically contains interveningsequences or introns that are not translated. Alternatively, since theamino acid sequence is known, a synthetic gene encoding the protein tobe sorted can be constructed by standard solid-phaseoligodeoxyribonucleotide synthesis methods, such as the phosphotriesteror phosphite triester methods. The sequence of the synthetic gene isdetermined by the genetic code, by which each naturally occurring aminoacid is specified by one or more codons. Additionally, for isomers orother variant metal binding proteins, if a portion of the isomer'sprotein sequence is known, but the gene or messenger RNA has not beenisolated, the amino acid sequence can be used to construct a degenerateset of probes according to the known degeneracy of the genetic code.General aspects of cloning are described, for example, in Sambrook etal., supra; in B. Perbal, “A Practical Guide to Molecular Cloning” (2ded., John Wiley & Sons, New York 1988), in Berger &. Kimmel, “Guide toMolecular Cloning Techniques” (Methods in Enzymology, vol. 152, AcademicPress, Inc., San Diego, 1987), and in D. V. Goeddel, ed., “GeneExpression Technology” (Methods in Enzymology, vol. 185, Academic Press,Inc., San Diego, 1991).

The present invention includes methods for producing the novel metalbinding proteins of the present invention. For example, a method forproducing a metal binding protein having an amino acid sequenceanalogous to at least one metal binding protein from a brine shrimp(Artemia) includes providing an expression system, producing a novelmetal binding protein using the expression system and purifying orisolating the novel metal binding proteins to obtain a metal bindingprotein of the present invention.

Expression systems can be systems such as traditional manufacturingplants. For example, organisms such as brine shrimp can be grown and thenovel metal binding proteins of the present invention purified orextracted from the tissues of the brine shrimp. Alternatively,biomanufacturing systems using genetically engineered organisms(produced as described herein) capable of producing the novel metalbinding proteins can be used to produce the novel metal bindingproteins. For example, bacteria containing a metal binding proteinexpression vector can be cultured on large or small scale (depending onthe particular need). The novel metal binding proteins can then bepurified from the bacterial broth and used in metal binding processes.

Therefore, a novel metal binding protein of the present invention can beproduced by expression of a nucleic acid sequence encoding a metalbinding protein in a modified organism or host cell. Such a nucleic acidsequence includes, for example, a MT gene such as [SEQ ID NO: 1] or asequence encoding a fragment or functional metal binding domain of a MTgene. The isolation of nucleic acid sequences or segments encoding metalbinding proteins of the present invention are described above. Onceisolated, these nucleic acid sequences are then incorporated into anexpression vector. This expression vector is then use to modify anorganism for producing the novel metal binding proteins of the presentinvention. Expression or production is typically under the control ofvarious control elements associated with the vector construct. Suchelements can include promoters, operators, enhancers and negativeregulatory elements which allow the user to regulate production of thenovel metal binding proteins of the present invention. The conditionsrequired for expression of cloned protein sequences in modifiedorganisms are well known in the art.

The expressed metal binding proteins are then purified using standardtechniques. Techniques for purification of cloned proteins are wellknown in the art and need not be detailed further here. One particularlysuitable method of purification is affinity chromatography employing animmobilized antibody to a metal binding protein. Other proteinpurification methods include chromatography on ion-exchange resins, gelelectrophoresis, isoelectric focusing, and gel filtration, among others.Alternatively, the metal binding proteins of the present invention canbe purified following their expression from modified organisms bymethods such as precipitation with reagents (e.g. ammonium sulfate orprotamine sulfate as well as other methods known in the art).

A further understanding of the present invention will be accorded tothose skilled in the art from a consideration of the followingnon-limiting Examples. These examples illustrate the cloning andexpression of exemplary metal binding proteins in accordance with theteachings of the present invention that are useful in the removal orrecovery of heavy metals from substrates.

It is emphasized that these examples are illustrative of the principlesand teachings of the present invention and are not intended to limit thescope of the invention to exemplary brine shrimp (Artemia) metal bindingproteins alone.

EXAMPLE 1

In accordance to the teachings of the present invention, the followingexemplary protocols illustrate methods useful in the production,purification and analysis of the novel metal binding proteins of thepresent invention.

Sample Preparation

As a preliminary step in the isolation of the novel metal bindingproteins, Artemia brine shrimp were grown in artificial seawater (AS)(422.7 mM NaCl, 7.24 mM KCl, 22.58 mM MgCl₂-6H₂O, 25.52 mM MgSO₄-7H₂O,1.33 mM CaCl₂-2H₂O and 0.476 mM NaHCO₃). Artemia cysts (2.5 g) wereincubated for 48 hours in 250 ml of AS supplemented w/antibiotics at 30°C., rotating at 125 rpm. After 24 hrs, phototropic Artemia werecollected, cultured for an additional 24 hrs and then collected by clothfiltration. The shrimp were weighed and if not used immediately, storedat −80° C.

The Artemia were then homogenized in homogenization buffer (HB) (10 mMTris-HCl (pH 8.0), 0.1 mM DTT, 0.5 mM PMSF and 10 μg/ml Soybean TrypsinInhibitor) and resuspended in HB at 4 ml/gm wet wt of shrimp. Thehomogenate was passed through a Yamato LH-21 homogenizer three times ata setting of 800 rpm, filtered through Miracloth (Calbiochem) and thefiltrate centrifuged in a Sorvall SA-600 rotor @ 14,300 rpm, 4° C. for30 min. The lipid layer on top of the supernatant was removed by vacuumaspiration and the lower supernatant layer collected and centrifuged ina Beckman 50.2TI rotor @ 40K rpm, 4° C. for 90 min. Again, the upperlipid layer was removed and the lower supernatant recentrifuged at 150K(150K sup). The 150K sup was then used immediately or stored at −80° C.If used immediately, this product was then subjected to gel filtrationas follows. The gel filtration studies verified the novel metal bindingproteins' ability to bind to heavy metals.

Gel Filtration Studies

The 150K sup was centrifuged in a Sorvall SA-600 rotor at 8,500 rpm and40° C. for 30 min. The resulting supernatant was then filtered through aHPLC certified 0.45 micron LC13 acrodisc filter (Gelman Sciences). A 20ml aliquot of filtered 150K sup was incubated at 40° C. for 20 min with2 μl of ¹⁰⁹Cd (0.066 μCi) to radiolabel the metal binding proteins. Thesample was then applied to a Sephadex G-50 molecular weight exclusioncolumn (2.6×94 cm) previously equilibrated with 50 mM Tris-HCl (pH 8.0)saturated with N₂. One molar DTT (2 μl) was added to fractions 60-100prior to sample loading in order to maintain reducing conditions in thefractions containing the low molecular weight metal binding proteins.The column was eluted with 50 mM Tris (pH 8.0) at a flow rate of 20ml/hr while monitoring the eluate at 280 nm. During the elution period,the buffer reservoir was continually purged with N₂. Samples used foramino acid analysis were not radiolabeled.

The ¹⁰⁹Cd content (CPM) of the column fractions was determined with anAuto-Logic gamma counter (ABBOTT Laboratories). Zinc content wasmeasured by Flame or Furnace Atomic Absorption Spectroscopy andexpressed as PPB zinc/fraction. Prior studies indicated that two classesof metal binding proteins were present, one class being a high molecularweight fraction. However, the majority of ¹⁰⁹Cd eluted with a lowmolecular weight class of zinc containing metal binding protein. Asshown in FIG. 1, radioactive metal binding protein had a elution peakcorresponding to that for Zinc (roughly, fraction #50). The proteinconcentration of the Sephadex G-50 fractions was determined with a BCATotal protein assay kit (Pierce) according to manufacturer's protocol.The protein concentration of the low molecular weight fractions wasdetermined using the enhanced protocol which has greater sensitivitythan the standard protocol. The distinct structural features of thenovel metal binding proteins of the present invention were thenidentified in the following studies.

Metal Binding Protein Characterization Studies

All chromatographic and molecular weight studies were performed toascertain structural features of the novel metal binding proteins. Allprotocols used were as described previously in B. Harpham, “Isolation ofMetal Binding Proteins From Artemia”, Master's Thesis, California StateUniversity, Long Beach Library, 1998. Using anion exchange and reversephase chromatography techniques well known in the art and described, forexample, in B. Harpham “Isolation of Metal Binding Proteins FromArtemia”, supra, metal binding proteins from Artemia were purified anddetermined to have molecular weights and amino acid sequence lengthunexpectedly lower than other known metal binding proteins. UnderSDS-PAGE conditions, Artemia metal binding proteins have molecularweight of about 5.8 kDa as compared to 6-7 kDa for metal bindingproteins from other mammalian species. Protein analysis of Artemia metalbinding proteins indicate a sequence length of 48 amino acids. Artemiaamino acid sequence was unexpectedly and significantly shorter in lengththan other known metal binding proteins, which range in length from 60to 68 amino acid residues.

Due to the unexpectedly distinct structural features of the novel metalbinding proteins, attempts at isolating nucleic acid sequences encodinga novel metal binding protein of the present invention using priormethodologies failed. Therefore, an alternative strategy was employed toisolate our MT gene encoding a novel metal binding protein.

EXAMPLE 2

In accordance with the teachings of the present invention, the followingteaches an exemplary strategy effective for cloning a novel metalbinding protein genes. The cloned metal binding protein gene and itssequence information are particularly useful for expression of metalbinding proteins useful in the methods of the present invention.

Prior attempts at cloning and obtaining a gene encoding a brine shrimp(Artemia) metal binding protein had been unsuccessful. Therefore, usingprotein purification techniques described, for example, in B. Harpham“Isolation of Metal Binding Proteins From Artemia”, supra, sequence datawas obtained for the N-terminal region of isoforms of Artemia metalbinding protein. Sequence data from four of the metal binding proteinisoforms indicated identical amino acid sequences through amino acidnumber ten. Using this sequence information, oligonucleotide primersunique to our novel metal binding proteins were constructed and used toisolate a gene encoding a novel metal binding protein as follows.

Cloning and Sequencing of a Gene Encoding Artemia Metal Binding Protein

Total RNA was isolated from 48 hour nauplii using the RNAzol method.Forty-eight hour nauplii samples were prepared as described above inExample 1. The PolyTract Procedure (Promega, Wis.) was then used toisolate mRNA from the total RNA samples. cDNA was generated from themRNA using SuperScript and 3′ RACE Kit procedures (Cat#18373, Gibco/BRL,WI) and then subjected to the following synthesis reaction.

cDNA synthesis reaction: Artemia mRNA 25 μl (500 ng) DEPC H₂O 30 μl 10μM AP  5 μl

The above mixture was incubated for 10 min @ 70° C., then placed on icefor 1-2 min. Volatilized liquid was collected by centrifugation for 10sec @ 10,000 rpm. The following were then added to the above RNAcocktail to produce a PCR solution:

10 × PCR Buffer 10 μl 25 mM MgCl₂ 10. μl 10 mM dNTP 5. μl 0.1 mM DTT 10μl

The above resulting PCR solution was then mixed and incubated at 42° C.for 5 min. Five (5) .mu.l of Superscript II RT was added and the mixtureincubated @ 42° C. for 50 min for cDNA synthesis. The reversetranscription reaction was terminated by incubating the solution for 15min at 70° C. 5 μl of RNase was then added and the solution incubatedfor 20 min at 37° C. The final solution containing Artemia cDNA was thenstored at −20° C. until used for PCR amplification as described below.

PCR Amplification of Artemia Metal Binding Protein Sequences

The initial PCR Primer Sequences used were as follows:

5′primer (N-terminal side) designated “MT-Not I” [SEQ ID NO:5] consisted of 5′-ACC TAT GCG GCC GCA AAT GGA CTG CTG CAA GAA C-3′(underlined nucleotides designate the Not I restriction site); and3′ primer (C-terminal side) designated as “dT-Spe I” was: [SEQ ID NO: 6]5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT A-3′; or [SEQ ID NO: 7]5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT C-3′; or [SEQ ID NO: 8]5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT G-3′.

The above 5′ and 3′ primers were then used in the followingamplification cocktail.

PCR Reaction Cocktail: 10 × PCR Buffer 5 μl 25 mM MgCl₂ 3 μl 10 mM dNTP1 μl 10 mM dT-Spel 1 μl 10 μM MT-Not I 1 μl

To the above PCR Reaction cocktail, a Gem 50 wax bead was added to thetube and the tube incubated at 80° C. for 2-3 minutes. Upon hardening ofthe wax at room temp for 10-15 min, the following were layered on top ofthe hardened wax:

Sterile H₂O 36.5 μl cDNA mixture 2 μl Taq Polymerase 0.5 μl

This final mixture was then subjected to the following PCR amplificationprogram.

PCR Program:

Initial denaturation for 3 min @ 95° C., followed by 29 cycles of:

94° C. for 1 min

49° C. for 1 min

72° C. for 1 min

72° C. for 10 min

Hold at 4° C.

Once amplified, the PCR product was verified for successfulamplification on a 1.2% agarose gel. The PCR product was then purifiedfor subsequent cloning using Qiagen QIAquick Gel Extraction (Qiagen,CA), double digested to form compatible ends and the purified usingQiagen QIAQuick PCR Purification protocol. The following primers whichcontain modifying restriction sites incorporated into their sequencewere used to subclone the purified PCR product containing brine shrimpArtemia metal binding protein gene sequences.

MT Nco I [SEQ ID NO: 9] (5′ primer containing an Nde I site):            Nde I     Nco I 5′-GCT ACA CAT ATG TCC ATG GAC TGC TGC AAGAAC-3′ MT Sal I [SEQ ID NO: 10] (3′ primer containing Sal I site):            Sal I 5′-ACG AAC GTC GAC GCC TTT TTT TTT TTT TTT A-3′

Using the MT Nco I and MT Sal I primers, with an annealing temperatureof 72.degree. C. for 1 min, the Artemia MT nucleotide sequence wasamplified and then subsequently subcloned into the pGEM3 vector betweenthe vector's Eco RI and Sal I sites. Once subcloned, the cloned metalbinding protein gene can then be easily modified or further processedfor use in expression, production or other methods requiring use of anisolated nucleic acid encoding a metal binding protein.

The entire coding sequence for MT gene was then determined using a LiCor4200 L DNA sequencer. Sequence comparison studies of the MT gene fromArtemia indicate it to have unexpectedly different sequence as comparedto other known metal binding protein genes. When Artemia MT genesequence was aligned with that of Equine and Human MT, homology wasobserved. The ability of the exemplary metal binding protein of thepresent invention to bind heavy metals was then confirmed in thefollowing studies.

EXAMPLE 3

The following teaches an exemplary study which can be performed on anyof the novel metal binding proteins of the present invention to aid ineither the verification of a protein as a metal binding protein or inthe identification of a protein as a novel metal binding protein. Forexample, the metal binding proteins of the present invention are capableof binding heavy metals such as zinc, cadmium and copper. The ability ofan isolated protein to bind heavy metals was described and detailed inthe disclosed transformation of E. coli with an exemplary MT of thepresent invention and shown, as indicated in FIG. 1. As describedpreviously, modified organisms useful for producing the novel metalbinding proteins of the present invention can be made following theteachings provided herein. An exemplary modified organism includes atransgenic tobacco plant which is particularly useful in the methodsdescribed herein. The following illustrates exemplary methods useful inproducing a modified organism of the present invention.

EXAMPLE 4 Transgenic Tobacco Plant

The cDNA for MT cloned into TOPO.CR2 vector is referred to as pART_(mt).The coding sequence for the MT was cloned into a pUC18 based plasmidcontaining the omega 5′ untranslated region of the TMV coat protein inframe with the multiple cloning site. (See FIG. 2). This wasaccomplished by amplification of the MT coding sequence frompART.sub.mt1 using PCR primers containing an Nco I restriction site onthe 5′ primer and a Sal I site on the 3′ primer. The PCR product andvector were each restricted with Nco I and Sal I and purified. The PCRproduct was then ligated into the vector using T4 DNA ligase. Theligation mixture was used to transform DH5α cells by electroporation. LBmedia was inoculated with individual colonies and grown overnight.Plasmid was isolated and sequenced to verify the presence and integrityof the MT coding sequence.

The Eco RI/Xba I cassette was removed and cloned into the correspondingsites on the plant expression vector pSS. The pSS vector contains theconstitutive CMV promotor and transcription terminator sequence in framewith the multiple cloning site. This insures that ligation of the EcoRI/Xba I cassette into the vector places the MT gene sequence in-framewith the CMV promotor. We refer to the construct as pSSmt. The pSSmt waspropagated in DH5α cells, isolated and sequenced to verify the presenceand integrity of the MT gene as described above.

MT Expression in Tobacco Leaves

A. tumefaciens were transformed with the cytosolic pSS.sub.mt constructby electroporation and grown overnight at 27° C. in YEB medium, pH 7.4,containing antibiotics. The cells were collected and resuspended ininduction medium (YEB, pH 5.8, antibiotics and 20 μM Acetosyringone) andgrown overnight at 27° C. The next morning the cells were collected bycentrifugation and resuspended in infiltration medium (MMA buffercontaining antibiotics and 200 .mu.M Acetosyringone) to an A.sub.600 of1.5 and incubated at room temperature for 2 hrs. Plant leaves weresubmerged in the bacterial suspension and placed in a vacuum dessicator.The leaves were infiltrated under a vacuum of 30-40 mbar. The leaveswere placed at room temperature for 72 hours then ground to a finepowder in liquid nitrogen and extracted with 10 mM Tris pH=8.0, 0.05 mMDTT, 1 mM PMSF. The solution was clarified by centrifugation at 30,000×gand the supernatant assayed for MT using a ¹⁰⁹Cd metal binding assay.Metal binding activity is clearly evident in the leaves containing thegene for Artemia MT.

Treatment Bound Cd (CPM) Buffer 747 Untreated Leaves 5052 InfiltratedLeaves I 12874 Infiltrated Leaves II 12763

Stable Transformation of Tobacco (Nicotiana Tabacum)

A suspension of A. tumefaciens transformed with pSSmt were grown asdescribed above. Leaves were cut into small pieces (without the centralvein) and transferred into sterile weck glasses containing 50-100 ml ofbacterial suspension (A₆₀₀˜1.0) and incubated at room temperature for 30minutes. The leaf pieces were then transferred onto sterile Whatman 3MMfilterpaper pre-wetted with sterile water in plastic petri dishes. Thedishes were sealed with saran wrap and incubated at 26-28° C. in thedark for two days. The leaf pieces were then washed with sterile watercontaining antibiotics and transferred onto MS II agar plates. Thepieces were incubated at 25° C. for 3-4 weeks with a 16 hr photoperiod.When shoots began to form, the shoots were removed and transferred ontoMS III agar plates and incubated at 25° C. with a 16 hr photoperioduntil roots began to form. The small plants were transferred into weckglasses containing MS III medium and incubated at 25° C. with a 16 hrphotoperiod for about two weeks. The young plants were then planted intosoil. Young leaves from the plants were collected and assayed for MTactivity as described above to determine the transgenic plants.

In closing it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principals of the invention.Other modifications may be employed which are within the scope of theinvention and accordingly, the present invention is not limited to thatprecisely as shown and described in the present specification.

1. A substantially purified metal binding protein having an amino acidsequence analogous to at least one metal binding protein from a brineshrimp (Artemia).
 2. The substantially purified metal binding protein ofclaim 1 including an amino acid sequence selected from the groupconsisting of: [SEQ ID NO: 2] (1) MET ASP CYS CYS LYS ASN GLY CYS THRCYS ALA PRO ASN CYS LYS CYS ALA LYS ASP CYS LYS CYS CYS LYS GLY CYS GLUCYS LYS SER ASN PRO GLU CYS LYS CYS GLU LYS ASN CYS SER CYS ASN SER CYSGLY CYS HIS STOP; [SEQ ID NO: 4] (2) MET ASP CYS CYS LYS ASN GLY CYS THRCYS ALA PRO ASN CYS LYS CYS ALA LYS ASP CYS LYS CYS; and

(3) sequences incorporating one or more conservative amino acidsubstitutions thereof.
 3. An isolated nucleic acid comprising DNA havingat least 80% sequence identity to a DNA molecule having the sequence ofnucleotide residues 1 to 66 of: [SEQ ID NO: 1] 5′-ATG GAC TGC TGC AAGAAC GGT TGC ACC TGT GCC CCA AAT TGC AAA TGT GCC AAA GAC TGC AAA TGC TGCAAA GGT TGT GAG TGC AAA AGC AAC CCA GAA TGC AAA TGT GAG AAG AAC TGT TCATGC AAC TCA TGT GGT TGT CAC TGA-3′.


4. The substantially purified metal binding protein of claim 1 on asupport.
 5. A vector including the nucleic acid sequence of claim
 3. 6.A metal binding composition comprising a substantially purified metalbinding protein having an amino acid sequence analogous to a metalbinding domain of a metal binding protein from a brine shrimp (Artemia).7. A method for reducing the concentration of a metal in a substrate,said method comprising the steps of: contacting a metal binding proteinhaving an amino acid sequence analogous to at least one metal bindingprotein from a brine shrimp (Artemia) with said substrate to bind saidmetal to said metal binding protein; and separating said bound metalfrom said substrate.
 8. The method of claim 7 wherein said substrate isa fluid.
 9. The method of claim 7 wherein said substrate is soil. 10.The method of claim 7 wherein said contacting step is under a hightemperature condition.
 11. A method for the removal of metal from metalcontaminated waste, said method comprising the steps of; contacting ametal binding protein having an amino acid sequence analogous to atleast one metal binding protein sequence from a brine shrimp (Artemia)with said metal contaminated waste to bind said metal binding protein tosaid metal in said metal contaminated waste; and separating said a boundmetal from said contacted metal contaminated waste.
 12. The method ofclaim 11 further comprising the additional step of: producing the metalbinding protein in a modified organism.
 13. The method of claim 7wherein said metal binding protein is coupled to a support.
 14. Themethod of claim 11 wherein said metal binding protein is coupled to asupport.
 15. A modified organism producing a metal binding proteinhaving an amino acid sequence substantially similar to SEQ ID NO: 2 andconservative amino acid substitutions thereof.
 16. The modified organismof claim 15 wherein said modified organism is a transgenic plant. 17.The modified organism of claim 15 wherein said modified organism is atransgenic shrimp.
 18. The modified organism of claim 15 wherein saidmodified organism is a bacteria.
 19. A method for producing at least onemetal binding protein having an amino acid sequence analogous to atleast one metal binding protein from a brine shrimp (Artemia) comprisingthe steps of: providing an expression system capable of producing atleast one metal binding protein; producing at least one metal bindingprotein having an amino acid sequence analogous to at least one metalbinding protein from a brine shrimp (Artemia) utilizing said expressionsystem; and isolating said at least one metal binding protein having anamino acid sequence analogous to at least one metal binding protein froma brine shrimp (Artemia) from said expression system.